The long range goals of this research are to understand the microbial mechanisms of activated macrophages and to devise methods for therapeutic manipulation of infections. The experimental model for these studies is in vitro infection of murine macrophages with the bacterium Listeria monocytogenes (Lm). In non-activated, J774 or bone marrow-derived macrophages, bacteria escape from vacuoles into cytoplasm shortly after they are internalized by phagocytosis. Macrophages activated with interferon-( plus lipopolysaccharide inhibit growth of internalized bacteria by preventing their escape from the vacuole. The rapid escape and its direct inhibition by the activated macrophage provide a functional setting for identification of essential microbicidal chemistries inside activated macrophages. The hypotheses to be tested are that escape of Lm into cytoplasm of activated macrophages is inhibited by altered membrane trafficking and delivery into the vacuole of reactive oxygen and reactive nitrogen intermediates that inhibit perforation; and that these activities are coordinated by the controlled recruitment of the GTPases Rab5a and Rac2. This research project will determine how activation changes macrophage vacuolar compartments, by applying quantitative microscopic methods for measuring intracellular chemistries. The timing of vacuole and phagosome maturation in activated and non-activated macrophages will be quantified using time-lapse, ratiometric fluorescence microscopy of live, Lm-infected macrophages. The association of various fluorescent organelle markers with vacuoles containing wild-type or mutant Lm or with phagosomes containing opsonized erythrocytes will be quantified. Markers will include chimeras of citrine (a variant of YFP) plus actin, Rab5a, Rab7, LAMP-1, 3 phosphoinositide-binding domains, Rac1, Rac2, inducible nitric oxide synthase (iNOS), and gp47 phox, a component of the phagocyte oxidase complex. Fluorescence microscopy will be used to localize cholesterol. Vacuoles perforated by Lm will be identified using new methods for detecting bacterial escape into cytoplasm. Reactive oxygen intermediate (ROI) and reactive nitrogen intermediate (RNI) generation in vacuoles will be localized relative to vacuole maturation, perforation and bacterial escape. To identify signaling complexes, fluorescence resonance energy transfer (FRET) microscopy will be used to localize activated Rac1 and Rac2, and to detect interactions between those proteins and iNOS, gp47phox and Rab GTPases on vacuoles. FRET stoichiometry will be applied to measure relative concentrations of fluorescent chimeras on phagosomes and Lm vacuoles. The contributions of Rab5a and Rac2 to phagosome maturation and prevention of Lm escape in activated macrophages will be measured in cells expressing mutant forms of those molecules, including dominant negative or constitutively active Rac1, Rac2 and Rab5a, and in cells depleted of Rac2 via small inhibitory RNAs. Consequent effects on the efficiency of Lm escape from vacuoles will be quantified.